During the past year, research in the Molecular Immunology Section has been focused on the mechanisms of recognition by human CD8+ T cell antigen-specific receptors (TCRs). Analysis of TCR recognition has been examined using T cell functional assays plus kinetic and thermodynamic binding assays, and crystallography. A particular emphasis of these studies has been on the mechanism by which TCRs cross-react on a panel of eight structurally distinct haptenated peptides presented by HLA-A2. The haptens were coupled to the lysine residue at position 5 of the Tax-5K peptide (LLFG[K-hapten]PVYV. The results demonstrate that 71% of the haptenated-peptide-induced CTL lines could cross-react on at least one other peptide, indicating the high degree of cross-reactivity of these TCRs. We compared the effects of HLA-A2 mutants with substitutions at known TCR contact sites for recognition by hapten-cross-reactive CTL. Recognition of the A2 mutants was remarkably similar whether they were presenting the immunizing or the cross-reactive peptide, indicating that similar amino acid contacts are made by the TCR during recognition of both complexes. Collectively, these results suggest that TCRs possess the molecular flexibility to accomodate very structurally diverse ligands while retaining conserved interactions with the surface of the MHC molecule. We have previously shown that surface mutations on HLA-A2 that disrupt TCR recognition may vary with the identity of the bound peptide, with the exception of Lys66, when mutated to alanine disrupts 93% of over 250 different T cell clones or lines, independent of which peptide is bound. A crystallographic structure of a peptide/HLA-A2 molecule with the K66A mutation indicates that this mutation exposes a negatively charged glutamate (Glu63) underneath Lys66. Concurrent replacement of Glu63 with Gln (E63Q/K66A)restores TCR binding and function for T cells specific for five different peptides presented by HLA-A2. Comparison of crystallographic structures of the Tax peptide bound by wild-type HLA-A2, K66A, and E63Q/K66A showed no conformational differences in the bound peptide bound by either of these HLA-A2 molecules, indicating that the effects of the mutations were not due to induced conformational changes in the bound peptide. Our findings are consistent with the notion that each TCR arrives at a unique solution of how to bind a peptide/MHC complex, and that these interactions are most strongly influenced by the chemical and structural features of the bound peptide. Future studies will focus on obtaining structural data for the Tax-specific A6 TCR bound to the Tax/E63Q/K66A complex to determine the precise molecular interactions that account for the ability of the E63Q mutation to "cure" the effects of the K66A mutation.